DEMONSTRATION OF EXOSITE I-DEPENDENT INTERACTIONS OF THROMBIN WITH HUMAN FACTOR-V AND FACTOR VA INVOLVING THE FACTOR VA HEAVY-CHAIN - ANALYSIS BY AFFINITY-CHROMATOGRAPHY EMPLOYING A NOVEL METHOD FOR ACTIVE-SITE-SELECTIVE IMMOBILIZATION OF SERINE PROTEINASES

In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, fac tor Va, and to release the activation fragments E and C1. For the iden tification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobil izing thrombin and other serine proteinases specifically (greater than or equal to 92%) through their active sites and used in affinity chro matography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation p athway from the 93% +/- 12% inhibition of the rate of activation corre lated with specific binding of hirudin(54-65) to this exosite. Chromat ography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically a nd with apparently higher affinity, in an interaction that was prevent ed by hirudin(54-65). The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and frag ment E had no detectable affinity. These results demonstrate a previou sly undescribed, exosite I-dependent interaction of thrombin with fact or Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombi n to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteo lytic activation of the protein cofactor.